- Email: [email protected]
Differential expression of connective tissue growth factor (CTGF) in inflammatory bowel disease (IBD)
talk between IL-6 and TGF-[3 pathways. Methods: Model intestinal epithelial cell fines, Caco2-BBE and T84 were used. Results: Using cell surface biotinylation, we show that TGF[3receptor type I1 is predominamly present in the hasolateral membrane of intestinal epithelial cells. Basolateral stimulation of epithelial ceils with TGF-[31 (10 ng/ml) induces a time dependent phosphorylation of SMAD-2 (detectable at 30 minutes, peak at 2 hours and decrease at 4 hours). Further, we demonstrate that SMAD-4 co-immunoprecipitates with SMAD-2 at two hours after stimulation with TGF-[31 and subsequently translocates to the nucleus suggesting that TGF-[31 effectively signals in intestinal epithelia. To analyze whether TGF-[31 plays a role in the negative regulation of IL-6 signaling, ceils were treated with TGF-[31 (10ng/ml) for various times prior to IL-6 (100 ng/ml) stimulation and phosphoSTAT-3 and ICAM-1 expression induced by IL-6 was determined by Western blot. We show that pre-treatment of cells with TGF-[31 is associated vath a down-regulation of IL-6-induced phospho-STAT-3 (TGF + IL-6: 78% and 70% inhibition 5 and 30 minutes post IL-6 stimulation respectively compared to IL-6 alone) and ICAM-1 expression induced by IL-6 (TGF + IL-6: 82% inhibition compared to IL-6 alone 8 hours post IL-6 stimulation). Furthermore, TGF-[31 pretreatment resulted in a complete inhibition of IL-6 induced ICAM1 promoter activity (fold increase compared to unstimulated cells firefly/renilla luciferase activity: 1L-6 4.5, "fGF + 1L-6 1, TGF alone 1). TGF-[3-mediated inhibition of 1L-6-induced ICAM-1 expression was reversed by transfection with dominant negative SMAD-2 constructs Conclusions: 1) TGF-[3 receptor type ll is predominantly located on basolateral membrane and receptor stimulation activates SMAD pathway, 2) TGF-[31 down-regulates IL-6-induced tyrosina phoshorylation of STAT-3 and ICAM-1 expression and 3) SMAD-2 is required for the down regulation of IL-6 signaling by TGF-[3. Collectively, our data demonstrate a crosstalk between TGF-[3 and Ik-6 and TGF-[3 may play a role in the negative regulation of 1L6 signaling in intestinal epithelial cells.
NF-kB (pILS),Gel, and G1R Exwwdon In Humans and Mice with and without Colitis (UC vs DSS) NF,.KB Human Mou,
(UC)
Colon.IlL
n,d.
(DSS)
9 n.d,
Gel Haman
Mouu
162
47
(UC)
(DES)
G1R Haman Moutm
(UC)
(Dee)
n.d
n.d.
Colon214 138 162 48 211 236 Co/k~ EN-NL 90 114 122 118 98 125 EN- Co//~ 132 238 240 328 176 175 Data expressed in energy unitsper pixel. EN, enteric nerves; n.d., not detected; NI., normal; UC, ulcerativecolitis
M1200 Acquired Microvascular Dysfunction in Inflammatory Bowel Disease (IBD) : Loss of Nitric Oxide Mediated Vasodilation Ossama A. Hatoum, David G. Binion, Mary F. Otterson, David D. Gutterman Background and Aims: IBD (Crohn's disease and ulcerative colitis) is characterized by refractory inflammatory ulceration and damage to the intestine. Mechanisms underlying the impaired mucosal healing are not defined. Because microvaseular dysfunction resulting in diminished vasodilatory capacity and tissue hypoperfusion ts associated with impaired wound healing we tested the hypothesis that IBD is associated with impaired endothelial mediated vasodilation in local gut microvessels. Methods: The response of isolated intestinal submucosal arterioles (100-200p.m), to ace@choline (Ach) was determined from IBD and control patients undergoing bowel resection. Diameter of isolated cannulated vessels was measured by videomicroscopy. Results: After constriction (30-50%) with endothelin-1, dilation to graded doses of Ach (10 L 10 ~M) was observed in control vessels (max dilation (MD) 82 - 2%, EDS0 = 1. lxi04, n = 34). Inhibition of NO synthase (k-NAME) reduced MD to 54_+ 6%, P<0.05 n = 7. Further reduction was observed after indomethacin (MD = 23 -+ 10%, n = 8, p<0.05 vs. control). Constriction to Ach was observed after endothelial denudation (51 _+ 10%, n = 6). Ach dilation was reduced in chronically inflamed IBD vessels (MD = 15 -+ 2%, ED50 = 3.7xl 0 7, n = 33, p = <0.01 compared to controls) but not in vessels from patients with diverticulitis, or uninvolved IBD vessels, which responded similarly to controls. NO inhibitors exerted no effect on the involved IBD microvessels, while indomethacin led to paradoxical constriction in response to Ach (-54-+9%, n = 6). Endothelial lining of the IBD and control m~cmvessels was intact as assessed by uptake of Dil-Ac-kDk and fluorescence microscopy. Dilation to papaverine was similar in all groups. Conclusions: Endothelial dependent vasodilation is profoundly disturbed in chronically inflamed 1BD gut microvessels, characterized by significantlyimpaired N O mediated vasorelaxation. IBD vessels rely heavily on cyelo-oxygenase mediated vasoddation, and are maximally dilated through prostaglsndin dependent mechanisms. Microvascular dysfunction in IBD is localized to areas of chronic inflammatory damage, and is not generalized to un-involved areas of bowel. Microvascular dysfunction in IBD may contribute to disease progression by limiting tissue perfusion, impairing mucosal healing and aggravating inflammation.
M1203 The Migratory Potential of Crohn's Disease Myofibroblasts Differs Significantly between Inflamed Mucosa, Strictures and Fistulae Johannes K.-H. Meier, Sandra N. Leeb, Wemer Falk, Juergen Schoelmerich, Gerhard Rogler Background: Regulation of the migration of colonic myofibroblasts (CMF) is an important mechanism during wound healing, fistulae formation or intestinal fibrosis in Crohn's disease (CD). The factors and mechanisms, which affect myoflbroblast migration in inflammatory bowel disease are not completely understood. Methods: Primary cultures of CMF were isolated from the mucosa of surgical specimens of patients with active CD (CD-CMF) and of control persons (control-CMF). CMF were also isolated from strictures (stricture-CMF) or fistulae (fistula-CMF) of p~/tients with CD. Migration assays of CMF were performed in the modified 48-wefi Boyden chamber and the number of the migrated CMF was determined by evahiating four high power fields (hpf) at a magnification of 400x. Results: Fibronectin in CMF conditioned medium stimulates migration of CMF. Therefore fibronectin and 24h or 48h conditioned medium of CMF were used to stimulate migration of CD-, stricture- and fistula-CMF. Migration of CD-CMF from inflamed mucosa was significantly reduced when compared to control-CMF (24h CD-CMF conditioned medium: 49 -+ 9 vs. 94 _+ 11 cells/hpf, p = 0.006). Interestingly, migration of flstula-CMF was sigmficamly reduced when compared to CD-CMF isolated from inflamed mucosa (24h CD-CMF conditioned medium: 19 -+ 3 vs. 40 -+ 4 cells/hpf, p = 0.0003), whereas migration ofstricture-CMF tended to be increased when compared to CD-CMF (24h CD-CMF conditioned medium: 49 _+ 8 vs. 40 "4" 4 cells/hpf). Similar results were obtained with 48h conditioned medium of CD-CMF and 24h or 48h conditioned medium of stricture- or fistula-CMF and fibronectin. Fibronectin (10-50 p,g/ml) induced migration of all CMF cultures. 25 p,g/ml fibronectin induced migration of 53 -+ 5 CD-CMF/hpf, 62 -+ 6 stricture-CMF/hpf and 23 _+ 2 fistulaCMFfapf (CD-CMF vs. fistula-CMF: p<0.0001). Conclusion: CD-CMF in general showed a significantly reduced migration when compared to control-CMF. CMF isolated from inflamed mucosa, smctures or fistulae of CD-patients differ in their migratory potential. The severely reduced migratory potential of fistula-CMF could be an explanation for impaired wound healing and fistula formation.
M1201 Decreased expression of syncoflin mRNA in colonic epithelial cells of bacteriachallenged germ-free mice and ulcerative colitis Kouhei Fuknshima, Yuji Funayama, Httoshi Ogawa, Ken-Ichi Takahashi, lwao Sasaki Background and Aims: Intestinal m~croorganisms play role in pathology of inflammatory bowel disease. Analysis of bacterial reconstitution process using germ-free mice provides new insights into bacteria-associated mucosal inflammation. The aims of the present study were to identify genes preferentially expressed in colonic epithehal cells and down-regulated by bacterial challenge and to investigate expression of human homologue in inflammatory bowel disease. Methods: Germ-free mice were orally given bacterial suspension prepared from conventional counterparts (bacterial reeonstitution). Small and large intestinal epithelia were obtained at 3, 7, 14, and 28 day and from untreated germ-free and specific pathogenfree mice. Epithelial gene expression was compared by differential display. Expression of the gene of interest was confirmed by northern blot and also evaluated in dextran sulfatesodium induced colitis used as an inflammatory control. Expression of the human homologue was investigated using epithelial RNA from 22 control, 22 ulcerative colitis, and 21 Crohn's disease patients by quantitative RT-PCR. To analyze inhibition mechanism of the gene T84 and Caco2 cells were stimulated with IL-lb, IL-6, lipopolysaceharide, and dexamethazone alone or in combination, then the gene expression was evaluated. Results: We identified a strongly down-regulated gene in the colon by bacterial reconstitution and determined its full sequence of eDNA. The gene was mouse counterpart of syncolfin. Syneollin mRNA reraarkablly decreased at 3 day, successively increased at 7, 14, and 28 day. In contrast, its mRNA expression was stable in dextran sulfate-sodium induced colitis. When expression of the human homologue was examined, syncollin mRNA was significantly less in ulcerative colitis but not Crohn's disease. We observed increase rather than decrease of syncolhn mRNA in vitro when combination of IL-lb or IL-6 and dexamethazone was used as stimulants. Conclusion: Decrease of syncollin mRNA is relatively unique for bacterial reconstitution and ulcerative colitis, suggesting that syncolhn is constituent in colonic epithelial cells-emeric flora interaction and bacteria-associated mucosal inflammation.
This study was supported by the Deutsche Forschungsgemeinschafi(SFB 585) and the BMBF Kompetenznetzwerk Chronisch entzuendliche Darmerkrankungen (Core facility Regensburg).
M1204 Differential Expression of Connective Tissue Growth Factor (CTGF) in Inflanunatory Bowel Disease (IBD) Fabio F. Di Mola, Pierluigi Di Sebastiano, Andrea Gardini, Paolo Innocenti, Arthur Zimmermann, Markus W. Buechler, Helmut Friess Background & Aims: A major clinical problem with 1BD is often to differentiate clearly between CD and UC, which is important for the planning of adequate medical and surgical treatment. In the present study CTGF, a novel peptide involved in fibrotic disorders, has been analyzed in CD and UC patients to evaluate whether it is possible to discriminate between the two disorders. Material & Methods: 25 normal human intestinal tissue samples were obtained through an organ donor program. CD tissues were obtained from 28 individuals undergoing partial intestinal resection due to complications of the disease. UC tissue samples were obtained from 16 patients undergoing coleetomy due to complications of the disease. The expression of CTGF was studied by Northern blot analysis. In situ hybridization was used to localize mRNA moieties in the tissue samples. Results: Northern blot analysis revealed an overage of 5-fold increase in CTGF mRNA expression in 89% (25/28) of CD tissue samples by comparison with normal controls (p<0.0001). In contrast, in UC samples CTGF mRNA levels were comparable to those of normal controls. However, UC tissue samples exhibited enhanced TGF-betal mRNA levels (4-fold; p<0.05). In situ hybridization in CD samples showed CTGF mRNA localized especially in fibroblasts within the submucosal layer and in some areas of intense damage in the proximity of the luminal surface. Interestingly, in one case with high CTGF expression at the time of surgery, the histopathological and clinical criteria indicated UC. However, one year later, the diagnosis was changed to
M1202 TGF-B down regulates IL-6 signaling in intestinal epithelial cells: Critical role of SMAD-2 BalIit Walia, Lixin Wang, Didier Merlin, Sbanthi V. Sitaraman Background and significance: IL-6 is a pro-inflammatory cytokine that plays an important role in the pathogenesis of inflammatory bowel disease. TGF-[3 is a multifunctional cytokine that signals through the SMAD pathway and is a potent negative regulator of mucosal inflammation in the intestine. The aim of the present study is to determine possible cross
A-329
AGA Abstracts
CD due to the clinical course of the disease, with fistula formation in the small bowel. Moreover, in another pauent with enhanced CTGF mRNA expression, clinical history and histopatbology were -highly suggestive of UC, but the diagnosis was finally reported to be CD due to the cfinical course of the disease. Conclusions: CTGE was differentially expressed in IBD with higher level of expression in CD meanwhile only low level of expression were present in UC. These results are of interest if we consider the importance of a correct diagnosis before surgery in 1BD, because the surgical approach is quite different in CD and UC pauents
inhibitor, PD98059 (100 mM). Ahematively, cells were transiently transfected with an adenoviral vector carrying a dominant negative Akt which overexpressed an inhibitor of Akt. or a control constitutive protein expressing vector. Collagen was assessed by Northern analysis or Western immunoblot. Results: iGF-[ stimulation resulted in protein tyrosine phosphorylation of a size consistent with IRS-1, IRS-2, Shc, and IGF-1R. Identity of the hands was confirmed by reblotting. Co-immunoprecipitation studies demonstrated a close assocaation between the receptor and the three early docking proteins. The MAP kinase inhibitor PD98059 resulted in a 31.7% reduction in IGF-I mediated increase in collagen al(1) mRNA (p<0.05; n = 3). Transfection with the dominant negative Akt resulted in a 55% percent reduction in IGF-I mediated increase in collagen type I protein (n = 2). Conclusions: These studies suggest that the MAPK pathway mediates IGF mediated collagen synthesis in intestinal smooth muscle.
M1205 High Producer of Lipoprotein(a) Show a Poor Prognosis in Patients with Crohn's Disease Mitsunori Noguchi, Shinichi Oikawa, Yoshitaka Kinouchi, Susumu Hara, Nobuo Hiwatashi, Shin]i Kumagai, Tooru Shimosegawa
M1208 A Role for Activated T Cells in Osteoporosis in Crohn Disease Nancy Wyzga, Jeffrey S. Hyams, Trudy Lerer, Patncia M. Davis, Anne M. Griffiths, Samuel Varghese, Francisco A. Sylvester
Background: Lipoprotein(a)
Purpose: Activated T cells are emerging as regulators of bone mass in chronic inflammatory disorders. We have previously reported a state of low bone turnover in newly diagnosed, untreated children with active Crohn disease. Since Crohn disease is associated with T cell activation (particularly of the CD4 + Thl phenotype), we reasoned that circulating activated T lymphocytes from patients with Crohn disease secrete factors that inhibit osteoblast and osteoclast activity. Methods: Blood samples were collected from newly diagnosed, untreated children with active Crohn disease and healthy age- and sex- matched controls. T lymphocytes were isolated with a paramagnetic cell sorting system (MACS, Mihenyt Biotec, Auburn, CA) and activated in vitro with anti-CD3 and anti-CD28 antibodies. We then measured levels of critical factors in bone remodeling, inchidmg receptor activator of nuclear factor KB (RANKL), osteoprmegerin (OPG), and interferon (IFN)-"/in conditioned medium and serum. T o examine the effects of activated T lymphocytes on bone ceils, we treated C57BL/6 mouse osteoclast and osteoblast precursors with 10% conditioned medium from activated mouse splenic T cells. Results: We studied 13 children with active Cruhn disease (6F, mean age 16.13 y) and 13 controls (6F, mean age 15.98). Serum levels of remodeling factors were similar in both groups (Table). However, activated T lymphocytes (n=4) secreted 140X more IFN-'y than RANKL into the medium (Crohn 28.7 _+ 6.2 ng/mL vs 20 + 4 pg/mL ; control 19.4 _+ 8.0 ng/mL vs 7.6 _+ 2 pg/mL, respectively). OPG was not detectable Activated T cells from patients with Crobn disease tended to secrete higher levels of IFN9y than controls. Actwated mouse T cells secreted comparable levels of IFN-'? into the conditioned medium than patients with Crohn disease. Both routine osteoclast and osteoblast development was inhibited by conditioned medium from activated T cells. The effect was IFN-~-dependent. Conclusions: Serum levels of RANKL and OPG are similar in patients with Crohn disease and healthy controls. However, circulating activated T cells secrete large quantities of IFN-% a CD4 + Thl cytokine. These levels of IFN-'y are sufficient to inhibit the development of osteodasts and osteoblasts in vitro, suggesting that activated T cells play a role in inducing low bone turnover in these patients.
M1206 Mononuclear Leukocyte Adhesion to Poly l:C-Treated Colon Smooth Muscle Cells Is Partly Dependent on the Signal Transducer and Activator of Transcription (Stat)-I Sudip K. Bandyopadhyay, Carol A De La Motte, Scott A Strong BACKGROUND: We previously reported that the inflamed mucosa of persons with inflammatory bowel disease (IBD) contains increased amounts of hyahironan (HA) that emanates from muscularis mucosae cells into the lamina propria congested with mononuclear leukocytes. In human mucosal smooth muscle cell (SMC) cultures, virus infection or poly I:C (viral mimic) treatment markedly up-regulates SMC adhesiveness for unactivated mononuclear cells through a novel mechanism that involves interaction of leukocyte-expressed CD44 with SMC-produced HA. Stat-1 is a transcription factor that plays an important role in many signalling pathways associated v,ath inflammation. Thus, we investigated the possible role of Stat-1 in the adhesive interaction that occurs between [eukocytes and poly I:C-induced mucosal SMCs. METHODS: SMC cultures were derived from the colonic mucosa of human surgical specimens as well as the large bowel of Star-1 knockout and control wild type mice. Electrophoretic mobility shift assays (EMSA) were used t o determine Stat-I-DNA binding activity in the cell extracts of control and poly I:C-stimulated mucosat SMCs, and Western blot analysis was employed to confirm tyrosine phosphorylation of Stat- 1. Binding of leukocytes to SMCs was measured with radiolabeled U937 cells. RESULTS: Activation of Stat-1 was observed after poly I:C treatment of M-SMCs as determined by EMSA using a 32P-fabeled specific probe, and supershift assays with a Stat-I specific antibody confirmed the activated band's identity A tyrosine-phosphorylated Stat-1 band was also observed by Western blot analysis in the cell extracts from poly l:C-treated, but not untreated mucosal SMC cuhutres. PoD, i:C treatment of SMCs isolated from wild type mice substantially increased the adhesion of U937 cells above unstimulated cell levels, whereas induced adhesion to poly I:C treated colon SMCs from Stat-1 null mice was attenuated. CONCLUSIONS: Poly l:C-treated SMCs express activated Stat-1 protein that likely regulates a hyaluronan-mediated interaction between these mucosa[ mesenchymal cells and recruited leukocytes.
Serum Cytoklne levels In Children with Crohn Dlseue and Controls Crohn dlsase He4dthyControl= p value
OPG (pg/mL) 89.6 ~ 10 77.0,5 0,3
IFN.y (pg/mL) Not detected Not detected N.A.
M1209 VEGF Expression and Microvessel Diameter as Predictive Factors for Sensitivity to Steroid m Patients with Ulcerative Colitis Norihiro Hanabata, Yoshihiro Sasaki, Tsuyotoshi Tsuji, Ry'ukzchi Hada, Akihiro Munakata Background: In ulcerative colitis (UC), the serum VEGF or the VEGF production of colonic mucosa have been reported to increase. While, VEGE expression has not yet been quantified in the colonic mucosa of UC. Aims: To quantify VEGF expression, microvessel counts and diameter and to correlate the parameters with the sensitivity to steroid therapy. Methods: Colorectal initial biopsy specimens from 39 UC with high sensitivity to steroid (H-UC), 9 UC with low sensitivity to steroid (DUC) and 6 normal controls (NC) were examined. Tissue sections were immunostained with anti-VEGF antibody for total inflammatory cell counts (/mm2), VEGF-positive cell counts (/mm 2) and VEGF-positive ratio (%), and with CD34 for microvessel counts (/ram 2) and the mean diameter (v.m). Results: H-UC had a significantly larger total cell (10048 -+ 2751, p < 0.0001) or VEGF-positive cell (2363 • 707, p<0.0001) than NC (7235 • 2088 or 1537 _+297, respectively) with no difference in VEGFpositive ratio (24.3• for H-UC vs. 22.7_+6.9 for NC) (Table). L-UC had a significantly lower VEGF-positive ceil (1420 _+701, p < 0.0005) or VEGF-posinve ratio (11.6 • 5.5, p< 00005) than H-UC While, microvessel counts were almost constant regardless of the groups (345+_70 for NC vs. 346_+99 for H-UC vs. 349_+114 for L-UC). Significant increase in microvessel diameter was seen comparing NC (6.68• to H-UC (7.83_+ 1.09, p< 0.0001) and H-UC to L-UC (9.05 -+ 120, p < 0.03). Out of the 5 parameters, VEGF-posnive ratio and microvessel diameter had a predictive value for L-UC with an 88.9% sensitivity and 88.9% specificity. Conclusions: DUC was characterized either as VEGF underexpression or enlarged microvesseL The disruption of healing process or disturbance of microcirculation may be involved in low sensitivity to steroid therapy in UC
M1207 IGF-I increases collagen synthesis by the MAPK pathway in rat intestinal smooth muscle Ellen M. Zimmermann, Xiping Xin, Phylissa Schmiedlin-Ren, Yong Tat Hou, Gregory M. Christman Insulin-like growth factor I (IGF-I) is a potent flbrogenic growth factor that stimulates proliferation of intestinal smooth muscle and increases smooth muscle cell (smc) collagen synthesis. These processes contribute to intestinal strictures that develop in patients with Crohn's disease. Little is known about the mechanisms involved in smc gene regulation in intestinal smooth muscle. The aim of this study was to determine the early docking proteins associated with 1GF-I receptor signal transduction rat intestinal smc. Methods: Primary cultures of rat intestinal smc were treated with 100 ng/ml IGF-1 in serum-free medium. Cell lysstes were prepared and immunoprecipitation performed using antibodies to IRS-1, IRS2, Shc and IGF-IR. Proteins were separated on 9% SDS-PAGE gels, transferred to nitrocellulose and detected by immunoblotttng using an ECL system. To determine the effect the MAP kinase pathway on 1GF-I mediated collagen synthesis, cells were treated with the MAP kinase
AGA Abstracts
RANKL (pg/mL) 9 ~6.6 12.2,3.8 0,68
A-330
NF-kB (pILS),Gel, and G1R Exwwdon In Humans and Mice with and without Colitis (UC vs DSS) NF,.KB Human Mou,
(UC)
Colon.IlL
n,d.
(DSS)
9 n.d,
Gel Haman
Mouu
162
47
(UC)
(DES)
G1R Haman Moutm
(UC)
(Dee)
n.d
n.d.
Colon214 138 162 48 211 236 Co/k~ EN-NL 90 114 122 118 98 125 EN- Co//~ 132 238 240 328 176 175 Data expressed in energy unitsper pixel. EN, enteric nerves; n.d., not detected; NI., normal; UC, ulcerativecolitis
M1200 Acquired Microvascular Dysfunction in Inflammatory Bowel Disease (IBD) : Loss of Nitric Oxide Mediated Vasodilation Ossama A. Hatoum, David G. Binion, Mary F. Otterson, David D. Gutterman Background and Aims: IBD (Crohn's disease and ulcerative colitis) is characterized by refractory inflammatory ulceration and damage to the intestine. Mechanisms underlying the impaired mucosal healing are not defined. Because microvaseular dysfunction resulting in diminished vasodilatory capacity and tissue hypoperfusion ts associated with impaired wound healing we tested the hypothesis that IBD is associated with impaired endothelial mediated vasodilation in local gut microvessels. Methods: The response of isolated intestinal submucosal arterioles (100-200p.m), to ace@choline (Ach) was determined from IBD and control patients undergoing bowel resection. Diameter of isolated cannulated vessels was measured by videomicroscopy. Results: After constriction (30-50%) with endothelin-1, dilation to graded doses of Ach (10 L 10 ~M) was observed in control vessels (max dilation (MD) 82 - 2%, EDS0 = 1. lxi04, n = 34). Inhibition of NO synthase (k-NAME) reduced MD to 54_+ 6%, P<0.05 n = 7. Further reduction was observed after indomethacin (MD = 23 -+ 10%, n = 8, p<0.05 vs. control). Constriction to Ach was observed after endothelial denudation (51 _+ 10%, n = 6). Ach dilation was reduced in chronically inflamed IBD vessels (MD = 15 -+ 2%, ED50 = 3.7xl 0 7, n = 33, p = <0.01 compared to controls) but not in vessels from patients with diverticulitis, or uninvolved IBD vessels, which responded similarly to controls. NO inhibitors exerted no effect on the involved IBD microvessels, while indomethacin led to paradoxical constriction in response to Ach (-54-+9%, n = 6). Endothelial lining of the IBD and control m~cmvessels was intact as assessed by uptake of Dil-Ac-kDk and fluorescence microscopy. Dilation to papaverine was similar in all groups. Conclusions: Endothelial dependent vasodilation is profoundly disturbed in chronically inflamed 1BD gut microvessels, characterized by significantlyimpaired N O mediated vasorelaxation. IBD vessels rely heavily on cyelo-oxygenase mediated vasoddation, and are maximally dilated through prostaglsndin dependent mechanisms. Microvascular dysfunction in IBD is localized to areas of chronic inflammatory damage, and is not generalized to un-involved areas of bowel. Microvascular dysfunction in IBD may contribute to disease progression by limiting tissue perfusion, impairing mucosal healing and aggravating inflammation.
M1203 The Migratory Potential of Crohn's Disease Myofibroblasts Differs Significantly between Inflamed Mucosa, Strictures and Fistulae Johannes K.-H. Meier, Sandra N. Leeb, Wemer Falk, Juergen Schoelmerich, Gerhard Rogler Background: Regulation of the migration of colonic myofibroblasts (CMF) is an important mechanism during wound healing, fistulae formation or intestinal fibrosis in Crohn's disease (CD). The factors and mechanisms, which affect myoflbroblast migration in inflammatory bowel disease are not completely understood. Methods: Primary cultures of CMF were isolated from the mucosa of surgical specimens of patients with active CD (CD-CMF) and of control persons (control-CMF). CMF were also isolated from strictures (stricture-CMF) or fistulae (fistula-CMF) of p~/tients with CD. Migration assays of CMF were performed in the modified 48-wefi Boyden chamber and the number of the migrated CMF was determined by evahiating four high power fields (hpf) at a magnification of 400x. Results: Fibronectin in CMF conditioned medium stimulates migration of CMF. Therefore fibronectin and 24h or 48h conditioned medium of CMF were used to stimulate migration of CD-, stricture- and fistula-CMF. Migration of CD-CMF from inflamed mucosa was significantly reduced when compared to control-CMF (24h CD-CMF conditioned medium: 49 -+ 9 vs. 94 _+ 11 cells/hpf, p = 0.006). Interestingly, migration of flstula-CMF was sigmficamly reduced when compared to CD-CMF isolated from inflamed mucosa (24h CD-CMF conditioned medium: 19 -+ 3 vs. 40 -+ 4 cells/hpf, p = 0.0003), whereas migration ofstricture-CMF tended to be increased when compared to CD-CMF (24h CD-CMF conditioned medium: 49 _+ 8 vs. 40 "4" 4 cells/hpf). Similar results were obtained with 48h conditioned medium of CD-CMF and 24h or 48h conditioned medium of stricture- or fistula-CMF and fibronectin. Fibronectin (10-50 p,g/ml) induced migration of all CMF cultures. 25 p,g/ml fibronectin induced migration of 53 -+ 5 CD-CMF/hpf, 62 -+ 6 stricture-CMF/hpf and 23 _+ 2 fistulaCMFfapf (CD-CMF vs. fistula-CMF: p<0.0001). Conclusion: CD-CMF in general showed a significantly reduced migration when compared to control-CMF. CMF isolated from inflamed mucosa, smctures or fistulae of CD-patients differ in their migratory potential. The severely reduced migratory potential of fistula-CMF could be an explanation for impaired wound healing and fistula formation.
M1201 Decreased expression of syncoflin mRNA in colonic epithelial cells of bacteriachallenged germ-free mice and ulcerative colitis Kouhei Fuknshima, Yuji Funayama, Httoshi Ogawa, Ken-Ichi Takahashi, lwao Sasaki Background and Aims: Intestinal m~croorganisms play role in pathology of inflammatory bowel disease. Analysis of bacterial reconstitution process using germ-free mice provides new insights into bacteria-associated mucosal inflammation. The aims of the present study were to identify genes preferentially expressed in colonic epithehal cells and down-regulated by bacterial challenge and to investigate expression of human homologue in inflammatory bowel disease. Methods: Germ-free mice were orally given bacterial suspension prepared from conventional counterparts (bacterial reeonstitution). Small and large intestinal epithelia were obtained at 3, 7, 14, and 28 day and from untreated germ-free and specific pathogenfree mice. Epithelial gene expression was compared by differential display. Expression of the gene of interest was confirmed by northern blot and also evaluated in dextran sulfatesodium induced colitis used as an inflammatory control. Expression of the human homologue was investigated using epithelial RNA from 22 control, 22 ulcerative colitis, and 21 Crohn's disease patients by quantitative RT-PCR. To analyze inhibition mechanism of the gene T84 and Caco2 cells were stimulated with IL-lb, IL-6, lipopolysaceharide, and dexamethazone alone or in combination, then the gene expression was evaluated. Results: We identified a strongly down-regulated gene in the colon by bacterial reconstitution and determined its full sequence of eDNA. The gene was mouse counterpart of syncolfin. Syneollin mRNA reraarkablly decreased at 3 day, successively increased at 7, 14, and 28 day. In contrast, its mRNA expression was stable in dextran sulfate-sodium induced colitis. When expression of the human homologue was examined, syncollin mRNA was significantly less in ulcerative colitis but not Crohn's disease. We observed increase rather than decrease of syncolhn mRNA in vitro when combination of IL-lb or IL-6 and dexamethazone was used as stimulants. Conclusion: Decrease of syncollin mRNA is relatively unique for bacterial reconstitution and ulcerative colitis, suggesting that syncolhn is constituent in colonic epithelial cells-emeric flora interaction and bacteria-associated mucosal inflammation.
This study was supported by the Deutsche Forschungsgemeinschafi(SFB 585) and the BMBF Kompetenznetzwerk Chronisch entzuendliche Darmerkrankungen (Core facility Regensburg).
M1204 Differential Expression of Connective Tissue Growth Factor (CTGF) in Inflanunatory Bowel Disease (IBD) Fabio F. Di Mola, Pierluigi Di Sebastiano, Andrea Gardini, Paolo Innocenti, Arthur Zimmermann, Markus W. Buechler, Helmut Friess Background & Aims: A major clinical problem with 1BD is often to differentiate clearly between CD and UC, which is important for the planning of adequate medical and surgical treatment. In the present study CTGF, a novel peptide involved in fibrotic disorders, has been analyzed in CD and UC patients to evaluate whether it is possible to discriminate between the two disorders. Material & Methods: 25 normal human intestinal tissue samples were obtained through an organ donor program. CD tissues were obtained from 28 individuals undergoing partial intestinal resection due to complications of the disease. UC tissue samples were obtained from 16 patients undergoing coleetomy due to complications of the disease. The expression of CTGF was studied by Northern blot analysis. In situ hybridization was used to localize mRNA moieties in the tissue samples. Results: Northern blot analysis revealed an overage of 5-fold increase in CTGF mRNA expression in 89% (25/28) of CD tissue samples by comparison with normal controls (p<0.0001). In contrast, in UC samples CTGF mRNA levels were comparable to those of normal controls. However, UC tissue samples exhibited enhanced TGF-betal mRNA levels (4-fold; p<0.05). In situ hybridization in CD samples showed CTGF mRNA localized especially in fibroblasts within the submucosal layer and in some areas of intense damage in the proximity of the luminal surface. Interestingly, in one case with high CTGF expression at the time of surgery, the histopathological and clinical criteria indicated UC. However, one year later, the diagnosis was changed to
M1202 TGF-B down regulates IL-6 signaling in intestinal epithelial cells: Critical role of SMAD-2 BalIit Walia, Lixin Wang, Didier Merlin, Sbanthi V. Sitaraman Background and significance: IL-6 is a pro-inflammatory cytokine that plays an important role in the pathogenesis of inflammatory bowel disease. TGF-[3 is a multifunctional cytokine that signals through the SMAD pathway and is a potent negative regulator of mucosal inflammation in the intestine. The aim of the present study is to determine possible cross
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AGA Abstracts
CD due to the clinical course of the disease, with fistula formation in the small bowel. Moreover, in another pauent with enhanced CTGF mRNA expression, clinical history and histopatbology were -highly suggestive of UC, but the diagnosis was finally reported to be CD due to the cfinical course of the disease. Conclusions: CTGE was differentially expressed in IBD with higher level of expression in CD meanwhile only low level of expression were present in UC. These results are of interest if we consider the importance of a correct diagnosis before surgery in 1BD, because the surgical approach is quite different in CD and UC pauents
inhibitor, PD98059 (100 mM). Ahematively, cells were transiently transfected with an adenoviral vector carrying a dominant negative Akt which overexpressed an inhibitor of Akt. or a control constitutive protein expressing vector. Collagen was assessed by Northern analysis or Western immunoblot. Results: iGF-[ stimulation resulted in protein tyrosine phosphorylation of a size consistent with IRS-1, IRS-2, Shc, and IGF-1R. Identity of the hands was confirmed by reblotting. Co-immunoprecipitation studies demonstrated a close assocaation between the receptor and the three early docking proteins. The MAP kinase inhibitor PD98059 resulted in a 31.7% reduction in IGF-I mediated increase in collagen al(1) mRNA (p<0.05; n = 3). Transfection with the dominant negative Akt resulted in a 55% percent reduction in IGF-I mediated increase in collagen type I protein (n = 2). Conclusions: These studies suggest that the MAPK pathway mediates IGF mediated collagen synthesis in intestinal smooth muscle.
M1205 High Producer of Lipoprotein(a) Show a Poor Prognosis in Patients with Crohn's Disease Mitsunori Noguchi, Shinichi Oikawa, Yoshitaka Kinouchi, Susumu Hara, Nobuo Hiwatashi, Shin]i Kumagai, Tooru Shimosegawa
M1208 A Role for Activated T Cells in Osteoporosis in Crohn Disease Nancy Wyzga, Jeffrey S. Hyams, Trudy Lerer, Patncia M. Davis, Anne M. Griffiths, Samuel Varghese, Francisco A. Sylvester
Background: Lipoprotein(a)
Purpose: Activated T cells are emerging as regulators of bone mass in chronic inflammatory disorders. We have previously reported a state of low bone turnover in newly diagnosed, untreated children with active Crohn disease. Since Crohn disease is associated with T cell activation (particularly of the CD4 + Thl phenotype), we reasoned that circulating activated T lymphocytes from patients with Crohn disease secrete factors that inhibit osteoblast and osteoclast activity. Methods: Blood samples were collected from newly diagnosed, untreated children with active Crohn disease and healthy age- and sex- matched controls. T lymphocytes were isolated with a paramagnetic cell sorting system (MACS, Mihenyt Biotec, Auburn, CA) and activated in vitro with anti-CD3 and anti-CD28 antibodies. We then measured levels of critical factors in bone remodeling, inchidmg receptor activator of nuclear factor KB (RANKL), osteoprmegerin (OPG), and interferon (IFN)-"/in conditioned medium and serum. T o examine the effects of activated T lymphocytes on bone ceils, we treated C57BL/6 mouse osteoclast and osteoblast precursors with 10% conditioned medium from activated mouse splenic T cells. Results: We studied 13 children with active Cruhn disease (6F, mean age 16.13 y) and 13 controls (6F, mean age 15.98). Serum levels of remodeling factors were similar in both groups (Table). However, activated T lymphocytes (n=4) secreted 140X more IFN-'y than RANKL into the medium (Crohn 28.7 _+ 6.2 ng/mL vs 20 + 4 pg/mL ; control 19.4 _+ 8.0 ng/mL vs 7.6 _+ 2 pg/mL, respectively). OPG was not detectable Activated T cells from patients with Crobn disease tended to secrete higher levels of IFN9y than controls. Actwated mouse T cells secreted comparable levels of IFN-'? into the conditioned medium than patients with Crohn disease. Both routine osteoclast and osteoblast development was inhibited by conditioned medium from activated T cells. The effect was IFN-~-dependent. Conclusions: Serum levels of RANKL and OPG are similar in patients with Crohn disease and healthy controls. However, circulating activated T cells secrete large quantities of IFN-% a CD4 + Thl cytokine. These levels of IFN-'y are sufficient to inhibit the development of osteodasts and osteoblasts in vitro, suggesting that activated T cells play a role in inducing low bone turnover in these patients.
M1206 Mononuclear Leukocyte Adhesion to Poly l:C-Treated Colon Smooth Muscle Cells Is Partly Dependent on the Signal Transducer and Activator of Transcription (Stat)-I Sudip K. Bandyopadhyay, Carol A De La Motte, Scott A Strong BACKGROUND: We previously reported that the inflamed mucosa of persons with inflammatory bowel disease (IBD) contains increased amounts of hyahironan (HA) that emanates from muscularis mucosae cells into the lamina propria congested with mononuclear leukocytes. In human mucosal smooth muscle cell (SMC) cultures, virus infection or poly I:C (viral mimic) treatment markedly up-regulates SMC adhesiveness for unactivated mononuclear cells through a novel mechanism that involves interaction of leukocyte-expressed CD44 with SMC-produced HA. Stat-1 is a transcription factor that plays an important role in many signalling pathways associated v,ath inflammation. Thus, we investigated the possible role of Stat-1 in the adhesive interaction that occurs between [eukocytes and poly I:C-induced mucosal SMCs. METHODS: SMC cultures were derived from the colonic mucosa of human surgical specimens as well as the large bowel of Star-1 knockout and control wild type mice. Electrophoretic mobility shift assays (EMSA) were used t o determine Stat-I-DNA binding activity in the cell extracts of control and poly I:C-stimulated mucosat SMCs, and Western blot analysis was employed to confirm tyrosine phosphorylation of Stat- 1. Binding of leukocytes to SMCs was measured with radiolabeled U937 cells. RESULTS: Activation of Stat-1 was observed after poly I:C treatment of M-SMCs as determined by EMSA using a 32P-fabeled specific probe, and supershift assays with a Stat-I specific antibody confirmed the activated band's identity A tyrosine-phosphorylated Stat-1 band was also observed by Western blot analysis in the cell extracts from poly l:C-treated, but not untreated mucosal SMC cuhutres. PoD, i:C treatment of SMCs isolated from wild type mice substantially increased the adhesion of U937 cells above unstimulated cell levels, whereas induced adhesion to poly I:C treated colon SMCs from Stat-1 null mice was attenuated. CONCLUSIONS: Poly l:C-treated SMCs express activated Stat-1 protein that likely regulates a hyaluronan-mediated interaction between these mucosa[ mesenchymal cells and recruited leukocytes.
Serum Cytoklne levels In Children with Crohn Dlseue and Controls Crohn dlsase He4dthyControl= p value
OPG (pg/mL) 89.6 ~ 10 77.0,5 0,3
IFN.y (pg/mL) Not detected Not detected N.A.
M1209 VEGF Expression and Microvessel Diameter as Predictive Factors for Sensitivity to Steroid m Patients with Ulcerative Colitis Norihiro Hanabata, Yoshihiro Sasaki, Tsuyotoshi Tsuji, Ry'ukzchi Hada, Akihiro Munakata Background: In ulcerative colitis (UC), the serum VEGF or the VEGF production of colonic mucosa have been reported to increase. While, VEGE expression has not yet been quantified in the colonic mucosa of UC. Aims: To quantify VEGF expression, microvessel counts and diameter and to correlate the parameters with the sensitivity to steroid therapy. Methods: Colorectal initial biopsy specimens from 39 UC with high sensitivity to steroid (H-UC), 9 UC with low sensitivity to steroid (DUC) and 6 normal controls (NC) were examined. Tissue sections were immunostained with anti-VEGF antibody for total inflammatory cell counts (/mm2), VEGF-positive cell counts (/mm 2) and VEGF-positive ratio (%), and with CD34 for microvessel counts (/ram 2) and the mean diameter (v.m). Results: H-UC had a significantly larger total cell (10048 -+ 2751, p < 0.0001) or VEGF-positive cell (2363 • 707, p<0.0001) than NC (7235 • 2088 or 1537 _+297, respectively) with no difference in VEGFpositive ratio (24.3• for H-UC vs. 22.7_+6.9 for NC) (Table). L-UC had a significantly lower VEGF-positive ceil (1420 _+701, p < 0.0005) or VEGF-posinve ratio (11.6 • 5.5, p< 00005) than H-UC While, microvessel counts were almost constant regardless of the groups (345+_70 for NC vs. 346_+99 for H-UC vs. 349_+114 for L-UC). Significant increase in microvessel diameter was seen comparing NC (6.68• to H-UC (7.83_+ 1.09, p< 0.0001) and H-UC to L-UC (9.05 -+ 120, p < 0.03). Out of the 5 parameters, VEGF-posnive ratio and microvessel diameter had a predictive value for L-UC with an 88.9% sensitivity and 88.9% specificity. Conclusions: DUC was characterized either as VEGF underexpression or enlarged microvesseL The disruption of healing process or disturbance of microcirculation may be involved in low sensitivity to steroid therapy in UC
M1207 IGF-I increases collagen synthesis by the MAPK pathway in rat intestinal smooth muscle Ellen M. Zimmermann, Xiping Xin, Phylissa Schmiedlin-Ren, Yong Tat Hou, Gregory M. Christman Insulin-like growth factor I (IGF-I) is a potent flbrogenic growth factor that stimulates proliferation of intestinal smooth muscle and increases smooth muscle cell (smc) collagen synthesis. These processes contribute to intestinal strictures that develop in patients with Crohn's disease. Little is known about the mechanisms involved in smc gene regulation in intestinal smooth muscle. The aim of this study was to determine the early docking proteins associated with 1GF-I receptor signal transduction rat intestinal smc. Methods: Primary cultures of rat intestinal smc were treated with 100 ng/ml IGF-1 in serum-free medium. Cell lysstes were prepared and immunoprecipitation performed using antibodies to IRS-1, IRS2, Shc and IGF-IR. Proteins were separated on 9% SDS-PAGE gels, transferred to nitrocellulose and detected by immunoblotttng using an ECL system. To determine the effect the MAP kinase pathway on 1GF-I mediated collagen synthesis, cells were treated with the MAP kinase
AGA Abstracts
RANKL (pg/mL) 9 ~6.6 12.2,3.8 0,68
A-330